The present invention relates to immunoassays for an analyte, such as a drug, hormone or an antigen, in a liquid sample, such as body fluid. More particularly, the present invention relates to a method for the detection of an analyte in a body fluid using anti-allotypic monoclonal antibodies as capture reagents or as detector reagents.
Many types of ligand-receptor assays have been used to detect the presence of various substances in body fluids such as urine or blood serum. These assays typically involve antigen-antibody reactions and synthetic conjugates comprising enzymatic, fluorescent, chemiluminescent, or radioactive labels. In most of these assays, there is a receptor (e.g. an antibody) which is specific for the selected ligand (e.g., analyte or antigen), and a means for detecting the presence and/or amount of the antigen-antibody reaction product. Most current tests are designed to make a quantitative determination, but in some circumstances all that is required is a positive/negative indication.
These immunoassays must be very sensitive because of the often small concentration of the ligand of interest in the test fluid. However, test samples of body fluids contain numerous components, some of which can interfere with the immunoassay. For example, endogenous immunoglobulins or complement proteins present in the sample may react with the test immunoglobulins, causing misleading results. Immunoglobulins or complement proteins present in the sample may react with capture antibodies, analyte-specific binding proteins (which can be primary antibodies) which are part of the immunoassay, thereby preventing attachment of the analyte to the binding protein, and/or attachment of the binding protein to the antibodies (which can either be labeled or attached to a solid phase). Additionally, complement proteins in the test sample may bind to analyte-antibody complexes formed during the test and prevent attachment of the binding proteins to the capture or labeled antibodies. When the assay results are read and interpreted, they may indicate an incorrectly high or low concentration of analyte, depending on the assay format used. Such incorrect results may lead a physician to improperly diagnose a disease state, or prescribe an incorrect course of treatment. Because of these problems, competitive immunoassays, sandwich immunoassays and other immunological detection methods utilize various techniques to reduce interferences. However, these techniques have not solved all of the problems encountered in these detection methods. It is an object of the present invention to provide an improved immunoassay having less sensitivity to endogenous components in the sample and greater accuracy and discrimination for analytes of interest.
The present invention provides rapid, sensitive immunoassay methods for detecting the presence of analytes in body fluids. The methods have high sensitivity and accuracy, and less sensitivity to the presence of endogenous immunoglobulins and complement proteins in the sample. Use of the present methods provide immunoassays which reproducibly yield reliable results.
The present invention provides a method for improving the accuracy of an immunoassay for determining the amount of analyte in a patient sample, in particular, samples from human patients. The present method improves the accuracy and specificity of the results obtained from the immunoassay by reducing or eliminating the assays"" sensitivity to endogenous immunoglobulins and complement proteins in the sample. The method comprises providing as a capture reagent or as a detectable label an antibody specific for an allotypic determinant on a primary binding protein used to detect the analyte of interest. The primary binding protein typically is not attached to a solid phase, whereas the capture antibody typically is attached to a solid phase. However, in some assay formats, the primary binding protein may be bound to an antigen on a solid phase and detected by a labeled antibody. The patient sample can be, for example, blood serum or plasma, urine, lymphatic fluid or cerebrospinal fluid.
In one embodiment, the method comprises (a) combining the patient sample with (i) a primary binding protein specific for the analyte, and (ii) a known amount of detectably labeled tracer molecules specific for said binding protein; (b) incubating the combination under conditions sufficient to permit the analyte and the tracer molecules to competitively bind to the binding protein; (c) contacting the combination with a solid phase having immobilized thereon an allotype-specific capture antibody specific for the primary binding protein allotype; (d) incubating the combination under conditions sufficient to permit the binding protein to bind with the capture antibody; (e) separating the solid phase from the sample; and (f) detecting the amount of labeled tracer molecules bound to the solid phase. In this method, the analyte competes with the labeled tracer molecule for binding sites on the binding protein. Therefore, the greater the concentration of analyte present in the sample, the fewer tracer molecules will bind to the binding protein. The resulting signal is inversely proportional to the amount of analyte in the sample. The amount of analyte present can be determined by comparing the results obtained to a standard curve.
In another embodiment, both the primary binding protein and the anti-allotypic capture antibody may be free in the sample. In this embodiment, the anti-allotypic capture antibody may be conjugated with a capturable species, e.g., biotin. The primary binding protein, the anti-allotypic antibody and a known amount of labeled tracer are combined with the sample. The tracer can be either the actual analyte or a fraction or analog thereof which has a comparable affinity for the binding protein. The mixture is incubated for a time sufficient to permit the analyte or tracer molecule to bind with the binding protein. The mixture then is contacted with a solid phase having immobilized thereon a capture component specific for the capturable species, e.g., where the capturable species is biotin, the capture component can be avidin or streptavidin. The solid phase then is separated from the sample, and the signal is measured as described above. The use of an affinity couple, such as avidin-biotin, to immobilize the capture monoclonal antibody can be utilized in any of the different assay formats described herein.
Variations of the basic assay are possible. For example, the primary binding protein can be added to the reaction mixture separate from the allotype specific antibody which is attached to a solid phase. Alternatively, the analyte analog, which could be the analyte itself, can be attached to a solid phase to compete with the analyte from the sample for complexing the primary binding protein, which, when reacted with the labeled allotype-specific antibody, will yield, after separation of the solid phase, a labeled complex which can be measured. Furthermore, in each of these methods, several reagents can be premixed. For example, the anti-allotype reagent can be premixed with the primary binding protein before reaction with the patient sample.
The method of the invention may be designed to exploit conventional sandwich assay formats or other immunoassay formats. For example, in one embodiment of a sandwich technique, the primary binding protein binds to the analyte of interest to form a complex. This complex then binds to the immobilized capture protein which, in this embodiment, comprises a second antibody specific for an allotypic determinant on the binding protein, to form an immobilized capture protein-primary binding protein-analyte complex, which is then reacted with a labeled antibody to form a xe2x80x9csandwichxe2x80x9d. The solid phase containing the bound complex then is removed from the sample, and the amount of label is determined.
The invention further comprises reagents for carrying out the method. In one aspect, the reagent comprises a solid phase having immobilized thereon anti-allotypic monoclonal antibodies specific for an allotypic determinant on an analyte-specific primary antibody.
In another aspect the invention comprises anti-allotypic monoclonal antibodies for use as capture or secondary antibodies in immunoassay methods.
The use of anti-allotypic monoclonal antibodies enables construction of a family of extremely sensitive assay systems which minimize interference by endogenous immunoglobulins and complement proteins present in the sample.